BCH 110C MIDTERM 2 Questions And Answers

BCH 110C MIDTERM 2 Questions And
Answers
10.1.tDesign/describetatstrategyttotclonetatDNAtfragmenttintotspecifictrestrictiontsitestoftatpl
asmid.tDNAtcloningt-tCORRECTttANSWER-
1.ttaketatchunktoftrecombinanttDNAtandtplugtittintotatplasmid
2.tputtthetplasmidtbacktintotthetbacteriatcelltsotthattittcantreplicate
3.lysetthetplasmidstout,tthentpurifytthemt=>tgivestyoutatpopulationtoftgenes/plasmids
4.tplacetthattpopulationtintathosttcellt=>ttheytwilltreplicatetandtallowtusttotstudyttheirtfunction.t
Fortthemttotreplicatetinthosttcell,twetneedttotaddtatpromotert->tRNAt->tprotein
5.tusetplasmidtamplificationttotseparatetandtusetthetspecifictplasmidstwetwant

10.10.tWhattistatSoutherntblot?tWhattistittusedtfor?t-tCORRECTttANSWER-
IttallowstfortthetdetectiontoftatspecifictDNAtsequencetintDNAtsamples.tIttcombinesttransfertof
telectrophoresist-separatedtDNAtfragments-

totatfiltertmembranetandtsubsequenttfragmenttdetectiontbytprobethybridization.

HOW:
•tDigeststoftgenomictDNAtaretseparatedtontatgelt•tThetseparatedtpiecestarettransferredttotfilt
ert(nitrocellulose)tbytdiffusion,tortmoretrecentlytbytelectrophoresingtthetDNAtontotthetfiltert•tT
hetfiltertistthenttreatedtwithtalkalittotdenaturetthetDNA,tresultingtssDNAtbindsttotthetfiltert•tAtla
beledtcDNAtprobetthattistcomplementaryttotthetDNAtoftinteresttistthentappliedttotthetfiltert•tAt
positivetbandtshouldtbetdetectabletwherethybridizationtbetweentthetprobetandtDNAtoccurre
d

10.2.tWhattkindtoftenzymetistatreversettranscriptase?t-tCORRECTttANSWER-
it'stantenzymetthattsynthesizestatcomplementarytDNAtchaintontantRNAttemplate.tBcttotuset
PCRtwetneedtDNA;twetcan'ttusetRNA.

-Thetsingle-
strandedtcDNAtmoleculestsynthesizedtbytthetRTtenzymetaretconvertedtbytDNAtpolymeras
etintotdouble-tstrandedtcDNAtmoleculestduringtthetPCRtreactiont(togethert'RT-
PCR').tThetamplifiedtDNAtcantthentbetusedtfortcloning.tEachtclonetobtainedtintthistwaytistcal
ledtatcDNAtclone.

10.3.tWhattistatcDNA?tHowtistittgenerated?t-tCORRECTttANSWER-
ittistComplementarytDNA.tIttistgeneratedtfromtcellulartmRNAtwhichtistextractedtandtconvert
edtintotDNAtbytthetReversettranscriptasetenzymet(RT)toftretroviruses.

,1.textracttRNAtfromttissuetandtpurifytit
2.toncetpurifiedtyoutneedttotseparatetmRNAt(whichtistalmosttexclusivelytPolytAttail)tfromtRN
A
3.tusetatprimerttotreversettranscriptaset(Olygo-dTtprimer)t=>tRNA-DNAthybrid
4.tgettridtoftRNAtusingtRNAsetH
5.tcoupletresultingtDNAtstrandtwithtPCRtusingtDNAtpolymerase

10.4.tHowtcantyoutproducetmilligramtamountstoftatplasmidtfromtjusttatfewtnanograms?tDesc
ribetthetsteps.t-tCORRECTttANSWER-plasmidst(smalltcirculartbacterialtreplicons)

Desirabletcharacteristicstoftatvector:tWEtNEED
-tstablytreplicatestinthostt-tneedstorigintoftreplicationt(ori)t-teasilytintroducedtintothostt-
tusuallytintotbacteria,tsometimestyeastt-tbyttransformationt(plasmid)tortinfectiont(virus)t-

tmarkerttotselecttcellstcontainingtvectort-tusuallytantibiotictresistancetgenet-

tgoodtsitestfortinsertingt"cloned"tDNA




HOW:
1)tBacteriattakestuptplasmidt
2)tPlasmidtincreasestintbacteria.t
3)tbacteriatmultiples
4)tbacterialtcoloniestwilltproduce
5)tpurifytplasmidtDNAtaftertthetcelltlysestandtwe'lltgettmoretplasmids.

10.5.tWhattistatpolymerasetchaintreactiont(PCR)?t-tCORRECTttANSWER-
PCRtistantintvitrottechniquetfortthetamplificationt(masstproduce)toftatspecifictDNAtsequencet
fromtatcomplextmixture.
t-

Ittistachievedtbytusingtoligonucleotidetprimers:ttypicallytshort,tsingletstrandedtoligonucleotid
estthattaretcomplementaryttotthetoutertregionstoftknowntsequence.tNeedt2tprimers,tonetthatt
goestonetwaytandtthetothertonetthattgoestthetothertway.
t-

ThetoligonucleotidestservetastprimerstfortthermostabletDNAtpolymerasetandtthetdenaturedt
strandstoftthetlargetDNAtfragmenttservestastthettemplate.
ITtCONTAINS:
-tTargettDNA
-2tPrimers
-ThermostabletDNAtpolymeraset(hasttotsurvivethighttemperatures-
ex.tTaqtDNAtpolymerase)
-Nucleotidest(dNTPs)
-Mg2+t(cofactortfortDNAtpolymerase)
*Temperaturetcyclingtwhichtincreasesttotdenaturet2xtstrandstandtdecreasesttotannealtprim
er.

, 10.6.tHowtcantthetPCRtproceduretbetusedttotamplifytandtclonetanytDNAtfragment?t-
tCORRECTttANSWER-

1.tTaket2xtstrandedtDNAtandtraisetthettempttotdenaturetitt=>t2tsingletstrandstwilltresult
2.twetcooltitttotannealtprimerstonteachtstrandt
3.twetaddtDNAtpolymerasetandtnucleotidesttotstartttranscribingtthetstrand
4.t2tdoubletDNAtstrandstaretformed

10.7.tWhytistDNAtpolymerasetfromtthermustaquaticustusedtintthetPCRtreaction?t-
tCORRECTttANSWER-

bctittistthermostable=tittcantsurvivethighttemperaturestwhichtaretnecessarytfortthetsynthesist
oftDNA

10.8.tWhattcantfusingtatproteintwithtat'tag'tbetusefultfor?t-tCORRECTttANSWER-
Theytaretoftentinsertedtin-
framettothelptintpurifyingtthetresultingtfusiontproteint(whentthetinsertedtDNAtistintthetsametre
adingtframetastthetinterruptedtgene)tproduct.

Oligohistidinet('His')ttagthastathightaffinitytfortdivalenttmetaltionstliketnickelt(Ni2+)t==>tPermit
stpurificationtbytnickeltaffinitytchromatograph

10.9.tWhattistthetprincipletoftDNAtSeparationtbytAgarosetGeltElectrophoresis?t-
tCORRECTttANSWER-

•tDNAtistnegativelytchargedtduettotthetphosphatestintitstbackbonetandtmovesttowardtthetpos
itivetpolet
t-SmalltDNAtpiecesthavetlittletfrictionaltdrag,tsottheytmovetrapidlyt

t-LargetDNAsthavetmoretfrictionaltdragtsottheirtmobilitytistslowert

•tDistributestDNAtaccordingttotsizet
t-Largesttneartthettopt-

t-Smallesttneartthetbottomt+

•tDNAtiststainedtwithtfluorescenttdyetthattintercalatestbetweentthetbasest(Ethidiumtbromide,
tSafe-GreenTM,tSafe-RedTM)




Ittist-tUsuallytusedttotsizetDNAt-tcantalsotmeasuretsupercoiling

11.1tDefinetexon,tintrontandtsplicing.t-tCORRECTttANSWER-
Exon:taretsequencestthattreachtthetcytoplasmtastparttoftmaturetmRNAt(andtcontaintthetcod
edtsequencetfortthetprotein).tTheytaretthetonlytonestdecoded,ttheytstaytin.

intron:taretnon-
codingtinterveningtsequencestthattinterrupttthetcodingtsequencetoftmosttmammaliantgenest
(andtcorrespondingtpre-mRNAs).tNottentirelytuseless

Splicing:tProcesstwheretatnewlytmadetprecursortmessengertRNAt(pre-
mRNA)ttranscripttisttransformedtintotatmaturetmessengertRNAt(mRNA).tIttworkstbytremovin
gtalltthetintronst(non-
codingtregionstoftRNA)tandtsplicingtbackttogethertexonst(codingtregions).tFortnuclear-